Project Summary Our long-term goal is to understand the mechanism of a class of protein molecules in the membranes of cells, which transport substances in or out of cells, such as the key nutrients amino acids and glucose. The knowledge regarding the mechanisms of these proteins in turn enable us to pursue the pathogenic mechanisms of certain disease processes and to develop therapies. Generally, what underlies the transporting process is a series of conformational changes of the transporter protein. The goal of this proposal is to apply our newly developed high-resolution fluorescence-polarization-microscope-based method to determine the energetics and dynamics of the conformational changes of a biomedically important transporter protein found in some pathogenic bacteria. Structural biology has yielded abundant protein structures, revealing the structural basis of protein functions. However, a full understanding of a protein molecule must include both its spatial and temporal characteristics. We thus need to go beyond studying the behaviors of a protein on a near-atomic scale in a static manner, and study it in a dynamic manner instead. However, the required experimental information about protein dynamics is often lacking, due to the absence of relatively general methods for reliably tracking rapid angstrom-scale conformational changes of a protein. Generally, such small changes can be reliably and quantitatively resolved only with such structural techniques as crystallography or Cryo-EM, which, unfortunately, lack time resolution. Conventional light microscopy, on the other hand, may be time-resolved but its spatial resolution had remained too low to resolve angstrom-scale protein conformational changes. Recently, we have successfully resolved protein conformational changes on millisecond-and-angstrom scales by examining anisotropy of a single fluorescent label attached to a chosen segment in an examined protein, which is known to adopt a unique orientation in each crystal structural state of the protein. With a state- of-the-art polarization microscope and analytic analyses, we have achieved an effective angle resolution of 5- 10. Over this range, a rotational motion of a protein molecule of an average size would cause a 1.7 - 3.5 change in the chord distance. Applying this method to the transporter protein, we will determine the energetics and kinetics of conformational changes that underlie its transporting function. Integrating the resulting dynamic information with structural information will ultimately yield an integrated, full four-dimensional mechanistic model that accounts for the behaviors of the transporter protein, at the precision and accuracy of the underlying measurements. Success of our proposed study will transform the way we investigate the dynamic mechanisms of membrane proteins including transporters, and accelerate the transition from the current, mostly static approach of structural biology to dynamic structural biology.